1. Technical Field
The invention concerns the field of cell culture technology. It concerns a method for producing proteins as well as a method to generate novel expression vectors and host cells for biopharmaceutical manufacturing. The invention further concerns pharmaceutical compositions and methods of treatment.
2. Background
The market for biopharmaceuticals for use in human therapy continues to grow at a high rate with 270 new biopharmaceuticals being evaluated in clinical studies and estimated sales of 30 billions in 2003 (Werner, 2004). Biopharmaceuticals can be produced from various host cell systems, including bacterial cells, yeast cells, insect cells, plant cells and mammalian cells including human-derived cell lines. Currently, an increasing number of biopharmaceuticals is produced from eukaryotic cells due to their ability to correctly process and modify human proteins. Successful and high yield production of biopharmaceuticals from these cells is thus crucial and depends highly on the characteristics of the recombinant monoclonal cell line used in the process. Therefore, there is an urgent need to generate new host cell systems with improved properties and to establish methods to culture producer cell lines with high specific productivities as a basis for high yield processes.
Early approaches focused on process design and reactor design. Now the main improvements are driven by media formulation development and genetically engineering of host cells. The most common industrial mammalian host cell systems for the production of biopharmaceuticals are immortalized Chinese hamster ovary (CHO) cell lines (Wurm, 2004).
Initial metabolic engineering strategies to improve mammalian production cell lines focused on their ability to grow in suspension in serum free media. Stable expression of transferrin and insulin-like growth factor 1 (IGF-1) in CHO-K1 cells resulted in a cell line able to proliferate under protein-free conditions (Pak et al., 1996). Further approaches to improve the production cell lines included the use of regulatory DNA elements on the transfection vectors aimed to target or create transcriptional hot spots. Regulatory elements such as S/MARs (Scaffold/matrix-associated regions) which effect chromatin structure and UCOEs (Ubiquitous chromatin opening elements) derived from house keeping genes were both shown to positively effect specific productivities of recombinant proteins produced from CHO cell lines (Barnes and Dickson, 2006).
As apoptosis has been shown to be the predominant cause of cell death in mammalian cell culture production processes (al-Rubeai and Singh, 1998) the effect of expression of anti-apoptotic genes in mammalian host cells on culture viability was thoroughly investigated. Most antiapoptosis engineering strategies are focused on the overexpression of anti-apoptotic genes of the bcl-2 family (e.g. bcl-1 or bcl-xL; (Kaufmann and Fussenegger, 2003). By increasing the cellular resistance to apoptotic stimuli during fermentation, such as nutrient depletion and waste byproduct accumulation, production processes with apoptosis engineered cell lines showed prolonged culture viability and in some cases an increase in product yield (Chiang and Sisk, 2005).
Since most biopharmaceutical products are proteins that are secreted from the cells during the production process, the secretory transport machinery of the production cell line is another interesting target for novel host cell engineering strategies.
Protein secretion is a complex multi-step mechanism: Proteins destined to be transported to the extracellular space or the outer plasma membrane are first co-translationally imported into the endoplasmic reticulum. From there, they are packed in lipid vesicles and transported to the Golgi apparatus and finally from the trans-Golgi network (TGN) to the plasma membrane where they are released into the culture medium (Seth et al., 2006).
The yield of any biopharmaceutical production process depends largely on the amount of protein product that the producing cells secrete per time when grown under process conditions. Many complex biochemical intracellular processes are necessary to synthesize and secrete a therapeutic protein from a eukaryotic cell. All these steps such as transcription, RNA transport, translation, post-translational modification and protein transport are tightly regulated in the wild-type host cell line and will impact on the specific productivity of any producer cell line derived from this host.
Many engineering approaches have employed the growing understanding of the molecular networks that drive processes such as transcription and translation to increase the yield of these steps in protein production. However, as for any multi-step production process, widening a bottle-neck during early steps of the process chain possibly creates bottle necks further downstream, especially post translation. Up to a certain threshold, the specific productivity of a production cell has been reported to correlate linearly with the level of product gene transcription (Barnes et al., 2007). Further enhancement of product expression at the mRNA level, however, may lead to an overload of the protein synthesis, folding or transport machinery, resulting in intracellular accumulation of the protein product. Indeed, this can be frequently observed in current manufacturing processes (FIG. 1).
Specific targeted engineering approaches aimed to address this problem and to efficiently improve the secretion of protein products from eukaryotic cells are hampered by the current lack of understanding of the complex regulatory network that drives the transport of proteins to the plasma membrane.
The first studies on engineering the intracellular transport of secreted therapeutic proteins were centered around the overexpression of molecular chaperones like binding protein BiP/GRP78, protein disulfide isomerase (PDI). Chaperones are cellular proteins hosted within the endoplasmic reticulum (ER) and assist the folding and assembly of newly synthesised proteins. In contrast to what could be expected, BiP overexpression in mammalian cells has been shown to reduce rather than increase the secretion of proteins it associates with (Domer and Kaufman, 1994). Likewise, PDI overexpression in CHO cells reduced the expression of a TNFR:FC fusion protein (Davis et al., 2000), whereas the specific production rate of an antibody was increased by 40% (Borth et al., 2005). A possible explanation for these surprising findings, that the increase of the cell's protein folding capacity creates a production bottle neck further downstream, is supported by a report describing ER to cis-Golgi transport problems for IFN-gamma production in a CHO cell line (Hooker et al., 1999).
Another recent approach to increase the secretion capacity of mammalian cells is the heterologous overexpression of the transcription factor X-box binding protein 1 (XBP-1). XBP-1 is one of the master-regulators in the differentiation of plasma cells, a specialized cell type optimized for high-level production and secretion of antibodies (Iwakoshi et al., 2003). XBP-1 regulates this process by binding to the so called ER stress responsive elements (ERSE) within the promoters of a wide spectrum of secretory pathway genes, resulting in (i) a physical expansion of the ER, (ii) increased mitochondrial mass and function, (iii) larger cell size and (iv) enhanced total protein synthesis (Shaffer et al., 2004).
Recently, attempts were described to increase protein secretion by overexpressing XBP-1 in non-plasma cells, especially production cell lines. In CHO-K1 cells, the production level of two reporter proteins (secreted alkaline phospatase (SEAP) and secreted alpha-amylase (SAMY)) was shown to increase after XBP-1 introduction in CHO-K1 cells. However, no effect could be demonstrated in transient studies with other cell lines such as HEK293, HeLa or HT-1080 cells (Tigges and Fussenegger, 2006). The patent application WO2004111194 by Ailor Eric claims the overexpression of XBP-1 or ATF6 for the generation of highly productive cell lines.
Notably, XBP-1 does not only regulate plasma cell differentiation but also plays an important role in the unfolded protein response (UPR) (Brewer and Hendershot, 2005). The UPR represents a complex signal transduction network activated by inhibition of protein folding in the endoplasmic reticulum (ER). The UPR coordinates adaptive responses to this stress situation, including induction of ER resident molecular chaperone and protein foldase expression to increase the protein folding capacity of the ER, induction of phospholipid synthesis, attenuation of general translation, and upregulation of ER-associated degradation to decrease the unfolded protein load of the ER. Upon severe or prolonged ER stress, the UPR ultimately induces apoptotic cell death (Schroder, 2006).
The process of terminal differentiation, such as the maturation from a lymphocyte to a plasma cell, is usually regarded an apoptosis-like program, during which the cell loses its proliferative capacity to give rise to a terminally differentiated secretory cell. In fact, nearly all cell types specifically designed for high-level protein secretion (e.g. glandular cells, pancreatic beta cells) are terminally differentiated, are not able to proliferate and have a limited life-span before ultimately undergoing programmed cell death (Chen-Kiang, 2003). Therefore, overexpressing XBP-1 as a regulator of both plasma cell differentiation and UPR, is potentially disadvantageous due to its inherent risk to inhibit proliferation and/or induce apoptosis.
Taken together, there is a need for improving the secretory capacity of host cells for recombinant protein production. This might even become more important in combination with novel transcription-enhancing technologies and in high-titer processes in order to prevent post-translational bottle necks and intracellular accumulation of the protein product (FIG. 1). However, at present, there are two major hurdles on the way to targeted manipulation of the secretory transport machinery: The still limited knowledge about the underlying regulatory mechanisms and the requirement to prevent a concomitant growth-inhibitory or apoptotic response of the producer cell.
The present invention describes a novel and surprising role for the ceramide transfer protein CERT in the transport of secreted proteins to the plasma membrane and furthermore provides a method to efficiently improve the production of proteins that are transported via the secretory pathway from eukaryotic cells.
CERT (also known as Goodpasture antigen-binding protein) is a cytosolic protein essential for the non-vesicular delivery of ceramide from its site of production at the endoplasmic reticulum (ER) to Golgi membranes, where conversion to sphingomyelin (SM) takes place (Hanada et al., 2003).
Two CERT isoforms exist: the more abundantly expressed, alternatively spliced form missing a 26-amino-acid, serine-rich region (SEQ ID NO. 10, 11) and the full-length 624 amino acid protein, designated CERTL (SEQ ID NO. 12, 13) (Raya et al., 2000). Both CERT isoforms possess a carboxyterminal steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain that is necessary and sufficient for ceramide binding and transport (Hanada et al., 2003). START domains are highly conserved from fly and worm to humans (FIG. 2). They are ˜210 amino acids in length and form a hydrophobic tunnel that accommodates a monomeric lipid (Alpy and Tomasetto, 2005; Soccio and Breslow, 2003). START domains are found in 15 mammalian proteins, with CERT being most closely related to the phosphatidylcholine transfer protein Pctp, which binds and shuttles phosphatidylcholine (PC) between membranes, and StarD10, a lipid transfer protein specific for PC and PE (Olayioye et al., 2005; Soccio and Breslow, 2003; Wirtz, 2006). In addition to the START domain, the CERT proteins further contain an aminoterminal PH domain with specificity for PI(4)P that is responsible for Golgi localization (Hanada et al., 2003; Levine and Munro, 2002) and a FFAT (SEQ ID NO: 29) motif (two phenylalanines in an acidic tract) that targets the protein to the ER via interaction with the ER resident transmembrane proteins VAP-A and VAP-B (Kawano et al., 2006; Loewen et al., 2003).
The fundamental role of CERT in lipid trafficking was demonstrated in the Chinese hamster ovary cell line LY-A, in which the expression of a mutant non-functional CERT protein impaired ceramide transport, thus resulting in reduced cellular levels of sphingomyelin (Hanada et al., 2003). Non-vesicular lipid transfer is thought to occur at so-called membrane contact sites (MCS), at which the ER comes into close apposition with other organelles (Levine and Loewen, 2006). CERT may thus shuttle a very short distance between ER and Golgi membranes, or perhaps contact both compartments simultaneously. When overexpressed, the START domain of CERT is sufficient for ceramide transfer to the Golgi apparatus (Kawano et al., 2006). However, under physiological conditions, both Golgi and ER targeting motifs are essential for CERT function. In LY-A cells, CERT was identified to contain a mutation within its PH domain (G67E), rendering the protein defective in PI(4)P binding (Hanada et al., 2003). The requirement for PI(4)P for CERT function is further supported by a recent report that PI4KIII-beta activity is necessary for efficient ceramide trafficking to the Golgi (Toth et al., 2006), the enzymatic activity of which is stimulated by protein kinase D (PKD).
PKD belongs to a subfamily of serine-/threonine-specific protein kinases (comprising PKD1/PKCμ, PKD2 and PKD3/PKCυ) and was recently identified to be of crucial importance for the regulation of protein transport from the Golgi membrane to the plasma membrane (reviewed in (Rykx et al., 2003; Wang, 2006)). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol (DAG; (Baron and Malhotra, 2002)), a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine.
The present invention shows that PKD phosphorylates CERT on serine 132 adjacent to the PH domain, whereby PI(4)P binding, Golgi targeting and ceramide transfer activity are negatively regulated. Furthermore, by transferring ceramide that is required for DAG production to Golgi membranes, CERT stimulates PKD activity, thus establishing a regulatory feedback-loop that ensures the maintenance of constitutive secretory transport.
Importantly, the data provided furthermore show that in different eukaryotic cell lines (COS7 and HEK293), introduction of the gene encoding CERT significantly enhances the secretion of a heterologous protein into the culture medium. This effect is even more pronounced when using a CERT mutant which cannot be phosphorylated by PKD. Deletion of the phosphorylation acceptor site within CERT interrupts the negative control of PKD on CERT, but leaving the positive feedback of CERT on PKD intact through the support of ceramide conversion to sphingomyelin and DAG. It can therefore be speculated that the secretion enhancing mechanism of the present invention can be exerted not only by wild type CERT but also by all mutants of CERT which uncouple CERT from the negative influence of PKD, including point mutations of the acceptor serine, deletions including this residue as well as mutation or deletion of the PKD docking site within CERT or even the START domain alone.
CERT belongs to the family of StAR-related Lipid Transfer proteins (Soccio and Breslow, 2003), which are characterized by their START domains for lipid binding. As the START domain of CERT has been demonstrated to be both required and sufficient for CERT action (Hanada et al., 2003), it is possible that the secretion-promoting effect of CERT could equally be observed when overexpressing another member of this protein family. This is especially likely for the closely related members of the PCTP-subfamily, comprising PCTP (SEQ ID NO. 26, 27), CERT/GPBP itself, StarD7 and StarD10. These proteins have distinct lipid-binding specificities and could equally impact on the function of organelles involved in the secretion of heterologous proteins.
Furthermore, expression of the related proteins STARD4 (SEQ ID NO. 20, 21) and STARD5 (SEQ ID NO. 22, 23), that are induced upon ER stress, may function to fulfill the increased demand of lipid transfer of cells during a production process.
The existence of START domain proteins in eukaryotic organisms from fly, worm and mouse to humans indicates that the basic mechanisms of lipid trafficking are conserved among the eukaryotic kingdom. It furthermore suggests, that the principle described in the present invention—that is increasing secretion by enforced expression of CERT—may well be applicable to all eukaryotic cells, including yeast.
In summary, the present invention provides a method for enhancing the secretory transport of proteins in eukaryotic cells by heterologous expression of CERT, CERT mutants or another member of the START protein family. This method is particularly useful for the generation of optimized host cell systems with enhanced production capacity for the expression and manufacture of recombinant protein products.
The method described in the present invention is advantageous in several respects:
First, we demonstrate heterologous expression of CERT to be a strategy to enhance recombinant protein production by increasing the secretory capacity of the host cell. Enhancing the specific productivity of producer cells translates into higher product yields in industrial protein production processes. With the current trend towards high-titer processes and more sophisticated expression enhancing technologies, post-translational bottle necks will become the evident rate-limiting steps in protein production and hence will draw increasing attention to secretion engineering approaches.
Second, the START domain of CERT is highly conserved in eukaryotes from C. elegans to humans. This strongly suggests that the method of the present invention can not only be used in mammalian host cell systems, but is equally applicable for protein production in all eukaryotic cells, including insect cells and yeast cells.
As a third important feature, CERT as a cytosolic factor is not part of the unfolded protein response and thus is not involved in a cellular stress response program which induces the shut-down of protein translation and—if not resolved—leads to cell cycle arrest or even apoptosis. In contrast, by playing an independent role in lipid trafficking, targeting CERT might confer enhanced protein secretion without concomitant induction of apoptosis. Thus, overexpressing CERT in producer host cells might be advantageous over XBP-1 based genetically engineering approaches.
Fourth, it is shown in the present invention that mutation of Ser132 of CERT impairs the phosphorylation of CERT by PKD which frees CERT from a negative regulatory influence. Meanwhile, the positive stimulation of PKD by CERT via DAG is left intact (FIG. 3A). This finding places CERT in the signalling pathway “upstream” of PKD, which has been published to be critically involved in the regulation of the late stages of secretory transport, namely the transport from the trans-Golgi network to the plasma membrane (Liljedahl et al., 2001). With regard to protein transport, this means, that CERT acts “downstream” of the ER which makes CERT the preferable target for manipulation compared to XBP-1 or specific ER-residing proteins (FIG. 3B).
Since CERT can impact even on the latest steps of the secretory pathway, it can be speculated that heterologous expression of CERT has the potential to enhance secretion without creating bottle necks further downstream. To our knowledge, CERT is currently the most downstream acting target for genetical engineering of the secretory pathway to enhance heterologous protein production.
Taken together, the impact of the lipid-transfer protein CERT on the secretory transport from ER to Golgi and from the Golgi apparatus to the plasma membrane, without the disadvantageous connection to a growth-inhibiting or apoptosis-inducing stress response make CERT, CERT mutants and other START family proteins very attractive and promising targets for genetic engineering approaches aiming to enhance the secretory capacity of eukaryotic cells.
3. Applicability
The targeted manipulation of CERT which is described in the present invention can be used for a broad range of applications. In particular, two basic approaches can be distinguished:
(i) Overexpression and/or enhancing the activity of CERT or a CERT derivative to increase the secretory transport capacity of a cell, or
(ii) reducing CERT activity and/or expression as a means of gene therapy in order to reduce cancer cell proliferation and/or invasion.
Applicability of CERT Overexpression
The described invention describes a method to generate improved eukaryotic host cells for the production of heterologous proteins by introducing the gene encoding CERT, CERT mutants or other proteins of the START protein family. This will enable to increase the protein yield in production processes based on eukaryotic cells. It will thereby reduce the cost of goods of such processes and at the same time reduce the number of batches that need to be produced to generate the material needed for research studies, diagnostics, clinical studies or market supply of a therapeutic protein. The invention will furthermore speed up drug development as often the generation of sufficient amounts of material for pre-clinical studies is a critical work package with regard to the timeline.
The invention can be used to increase the property of all eukaryotic cells used for the generation of one or several specific proteins for either diagnostic purposes, research purposes (target identification, lead identification, lead optimization) or manufacturing of therapeutic proteins either on the market or in clinical development.
As shown in the present invention, heterologous expression of CERT does not only enhance protein secretion, but also has an influence on the abundance of transmembrane proteins on the cell surface. Inhibition or reduced expression of CERT leads to a dramatic reduction of the amount of cell surface receptors such as the transferrin receptor (FIG. 8). As secreted and transmembrane proteins share the same secretory pathways and are equally transported in lipid-vesicles, these data underscore the importance of CERT in the modulation of secretion as well as the transport of membrane-bound cell-surface receptors.
Therefore, the method described herein can also be used for academic and industrial research purposes which aim to characterize the function of cell-surface receptors. E.g. it can be used for the production and subsequent purification, crystallization and/or analysis of surface proteins. This is of crucial importance for the development of new human drug therapies as cell-surface receptors are a predominant class of drug targets. Moreover, it might be advantageous for the study of intracellular signalling complexes associated with cell-surface receptors or the analysis of cell-cell-communication which is mediated in part by the interaction of soluble growth factors with their corresponding receptors on the same or another cell.
Applicability of Decreasing/Inhibiting CERT
In the present invention, we provide evidence that the reduction of CERT expression leads to reduced secretion of soluble extracellular proteins as well as a lower abundance of cell surface receptors. This makes CERT an attractive target for therapeutic manipulation.
One of the hallmarks in the conversion from a normal healthy cell to a cancer cell is the acquisition of independency from the presence of exogenous growth factors (Hanahan and Weinberg, 2000). In contrast to the normal cell, tumor cells are able to produce all growth factors necessary for their survival and proliferation by themselves. In addition to this autocrine mechanism, cancer cells often show an upregulated expression of growth factor receptors on their surface, which leads to an increased responsiveness towards paracrine-acting growth and survival factors secreted from cells in the surrounding tissue. By targeting CERT in tumor cells, e.g. by using siRNA approaches, it might be possible to disrupt autocrine as well as paracrine growth-stimulatory and/or survival mechanisms in two ways: (i) By reducing growth factor transport and secretion and (ii) by decreasing the amount of the corresponding growth factor-receptor on tumor cells. Thereby both, the amount of growth stimulating signal and the ability of the cancer cell to perceive and respond to these signals will be reduced. Inhibition of CERT expression in cancer cells might therefore represent a powerful tool to prevent cancer cell proliferation and survival.
CERT might furthermore be a potent therapeutic target to suppress tumor invasion and metastasis. During the later stages of most types of human cancer, primary tumors spawn pioneer cells that move out, invade adjacent tissues, and travel to distant sites where they may succeed in founding new colonies, known as metastasis.
As a prerequisite for tissue invasion, cancer cells express a whole set of proteases which enable them to migrate through the surrounding healthy tissue, to cross the basal membrane, to get into the blood stream and to finally invade the tissue of destination.
Some of these proteases are expressed as membrane-bound proteins, e.g. MT-MMPs (Egeblad and Werb, 2002) and ADAMs (Blobel, 2005). Due to their crucial role in matrix remodelling, shedding of growth factors and tumor invasion, proteases themselves are discussed as drug targets for cancer therapy (Overall and Kleifeld, 2006). We hypothesize that inhibition of CERT expression and/or activity in tumor cells will reduce the amount of membrane-bound proteases on the surface of the targeted cell. This might decrease or even impair the invasive capacity of the tumor cell as well as its ability for growth factor shedding, resulting in reduced invasiveness and metastatic potential of the tumor. Thus, targeting CERT might offer a novel way of preventing late-stage tumorgenesis, especially the conversion from a benign/solid nodule to an aggressive, metastasizing tumor.
For therapeutic applications it is, thus, the goal to reduce and/or inhibit the activity and/or expression of CERT. This can be achieved either by a nucleotide composition which is used as human therapeutic to treat a disease by inhibiting CERT function whereby the drug is composed of an RNAi, and siRNA or an antisense RNA specificly inhibiting CERT through binding a sequence motive of CERT RNA. Reduction/inhibition of CERT activity/expression can also be achieved by a drug substance containing nucleotides binding and silencing the promoter of the CERT gene.
Furthermore, a drug substance or product can be composed of a new chemical entity or peptide or protein inhibiting CERT expression or activity. In case of a protein being the active pharmaceutical compound it may be a (i) protein binding to CERT promoter thereby inhibiting CERT expression, (ii) protein binding to CERT or PKD thus preventing binding of PKD and CERT and hindering CERT phosphorylation by PKD, (iii) a protein similar to CERT which however does not fulfill CERT functions, that means a “dominant-negative” CERT variant, or (iv) a protein acting as scaffold for both CERT and PKD, resulting in irreversible binding of CERT to PKD (=a stable PKD/CERT complex) which is not functional due to the inhibitory phosphorylation of CERT by PKD and the hindering of dissociation of CERT from said complex.